In vivo biosensors for small molecules remain a valuable means through which one can design regulatory networks, build screens/selections, and quantify in vivo concentrations of metabolites of interest following system perturbations. Transcription activator and repressor proteins—proteins that modulate the activity of the RNA polymerase on a given promoter—serve as perhaps the most straight forward and controllable option for an in vivo biosensor. Further, transcription repressor/activator proteins are numerous in nature and there exist a wide range of low molecular weight ligands to which they respond. The value of an in vivo biosensor is intrinsically founded upon its transcription profile. An ideal in vivo biosensor would possess little-to-no basal level of expression in the absence of an input signal and the output signal would be linear over a large dynamic range.
Putatively, σ54-dependent promoters and their associated activator proteins are ideally suited for use as in vivo biosensors. The sigma subunits of RNA polymerase specifically bind to DNA sequence elements and are responsible for differential gene expression. The primary, and most well understood, sigma factor is σ70. σ70 associates with the core RNA polymerase (E) to transcribe housekeeping genes. The complex E-σ70 alone can be sufficient to catalyze the open promoter complex and allow RNA transcription. While activity can be controlled by various repressor proteins, leaky expression persists.
Absolute control, however, is accomplished through the use the much less common complex E-σ54. While most bacteria contain several alternative σ factors of the σ70 class, usually only one σ54 form exists. A σ54-governed promoter is unique in that hydrolysis of the promoter DNA by an activator protein is an absolute requisite for transcription, imparting an intrinsically very low level of basal expression. ATP hydrolysis by activator proteins can be triggered by phosphorylation, binding of low molecular weight ligands, or protein-protein interaction. Further, without the need for a repressor protein, transcription levels can be tightly controlled over a large dynamic range. Lastly, the σ54-based transcription system is a dedicated transcription system with little cross-talk; there exist close to 100 σ54 molecules of in E. coli (compared to 700 of σ70), while there are only 20 σ54-governed promoters.
Use of σ54-dependent promoters and their cognate activator proteins as biosensors in E. coli has been limited to date. Most of the 20 native E. coli σ54-dependent promoters are induced in response to nitrogen limitation, potentially minimizing their use in biotechnology applications; however, prpBCDE promoters from Salmonella enteric serovar Typhimurium and E. coli have been explored by Lee, et al. as a propionate-inducible expression system (Lee, S. K., Keasling, J. D. A Propionate-Inducible Expression System for Enteric Bacteria. Appl. Environ. Microbiol. 71, 6856-6862 (2005)).